The present invention relates to methioninase expression systems containing two or more tandem nucleotide sequences that encode methioninase and to methods to produce recombinant methioninase from them. This system of production results in high amounts of methioninase activity. The methioninase thus produced is useful in antimethionine and antihomocysteine therapy.
Therapeutic drug-based treatment of cancer is directed at the use of medicinals which selectively inhibit or kill the cancer cells while not harming normal tissue function beyond acceptable amounts. The difficulty with conventional chemotherapy has been the toxicity of therapeutic drugs for normal tissue.
Many tumors have been shown to have absolute requirement for methionine in a variety of cell types and evaluated tumor tissues, including tumors of the colon, breast prostate, ovary, kidney, larynx melanoma, sarcoma, lung, brain, stomach and bladder as well as leukemias and lymphomas. Methionine dependence has been defined as an inability of tumors to grow when methionine is replaced by homocysteine in the growth medium. See, for example, Chello et al., Cancer Res, 33:1898-1904, 1973; and Hoffman, Anticancer Res, 5:1-30, 1985.
Methionine depletion has been shown to synchronize selectively methionine-dependent tumor cells into late S/G2 phase of the cell cycle. Hoffman et al, Proc Natl Acad Sci USA, 77:7306-7310, 1980. Using antimethionine chemotherapy which is methionine deprivation, followed by depletion of methionine and coupled with exposure to an antimitotic agent, tumor cells have been selectively eliminated from cocultures of normal and tumor cells, resulting in cultures of normal cells proliferating vigorously. Stem et al., J Natl Cancer Inst, 76:629-639, 1986.
In order for methionine-dependent chemotherapy to be conducted in vivo, it is necessary to have a means to effectively deplete serum of circulating methionine. Methionine depletion methods have not been described that reduce circulating methionine levels in vivo in a manner sufficient to be effective in antitumor therapies.
Methioninase, an enzyme which degrades methionine, has been purified from a variety of bacterial sources, and has been reported to slow the rate of tumor cell proliferation in vitro. Kreis et al., Cancer Res, 33:1862-1865, and 1866-1869, 1973; Tanaka et al., FEBS Letters, 66:307-311 1976; Ito et al., J Biochem 79:1263-1272, 1976; and Nakayama et al., Agric Biol Chem 48:2367-2369, 1984.
Kreis et al., Cancer Res 33:1866-1869, 1973, have described the use of highly impure methioninase preparations isolated from Clostridium sporogenes at 1150 units/kg/day to inhibit growth of carcinosarcoma cells implanted in a mouse model. Although the enzyme apparently reduced primary tumor cell growth, it was not reported to reduce the T/C (treated versus control) ratio of tumor diameter below 50%, and was not reported to have any effect on metastasis. The authors also indicated that tumor specificity of the methioninase cannot be expected without other unspecified interventions, and further do not comment on the possibility that endotoxin, or other components of the impure preparation, were responsible for the effects observed. The only toxicity studies reported were absence of animal body weight loss after the duration of the treatment, and negative gross examination for toxicity. Further, the authors report that the enzyme had a serum half life of 4 hours. Kreis et al., Cancer Res 33:1866-1869, 1973, further reported the use of a methionine-free diet as a means to deplete methionine as an antitumor therapy, but the diet did not slow tumor growth as effectively as the use even of an impure preparation of methioninase and resulted in the undesirable side effect of continuous loss of weight of the animal.
The parent applications herein disclose effective chemotherapy of tumors directed at reducing the amount of methionine as to provide a beneficial antitumor effect without deleterious injury using methioninase. The present invention improves the disclosed therapeutic and diagnostic methods and composition by providing a method to produce commercially viable quantities of highly pure recombinant methioninase, using expression systems containing at least two copies of the methioninase gene.
The present invention is based, in part, on the generation of methioninase expression systems containing two or more tandem copies of methioninase encoding nucleotide sequences. The expression systems of the present invention can produce recombinant methioninase in an appropriate host cell, such as E. coli, at levels ranging from about 40-75% of total cellular protein.
In a preferred embodiment methioninase expression systems containing two or more tandem copies of the P. putida methioninase gene operably linked to a T7 RNA polymerase promoter are described. These systems have been used to produce recombinant methioninase at about 1 to 4 gram/liter, with an activity of about 6.4 to about 12.4 units/mL and a specific activity of about 3.8 to about 10.2 units/mg before purification, using appropriate incubation conditions and purification methods.
The invention further provides methods of producing recombinant methioninase using cells containing the methioninase expression system of the present invention.
Substantially pure recombinant methioninase produced using cells containing the methioninase expression systems of the present invention is useful in compositions for diagnostic and therapeutic use, particularly in methods for inhibiting tumor cell growth to lower homocysteine levels in patients to reduce the risk of, and to treat, cardiovascular diseases, obesity and negative symptomologies of aging, as well as to deplete methionine for tumor diagnosis and imaging.
The recombinant methioninase may be provided in chemically modified forms, for example by coupling to polymers such as polyethylene glycol (PEG).